Dual‐contrast micro‐CT enables cartilage lesion detection and tissue condition evaluation ex vivo

Summary Background Post‐traumatic osteoarthritis is a frequent joint disease in the horse. Currently, equine medicine lacks effective methods to diagnose the severity of chondral defects after an injury. Objectives To investigate the capability of dual‐contrast‐enhanced computed tomography (dual‐CECT) for detection of chondral lesions and evaluation of the severity of articular cartilage degeneration in the equine carpus ex vivo. Study design Pre‐clinical experimental study. Methods In nine Shetland ponies, blunt and sharp grooves were randomly created (in vivo) in the cartilage of radiocarpal and middle carpal joints. The contralateral joint served as control. The ponies were subjected to an 8‐week exercise protocol and euthanised 39 weeks after surgery. CECT scanning (ex vivo) of the joints was performed using a micro‐CT scanner 1 hour after an intra‐articular injection of a dual‐contrast agent. The dual‐contrast agent consisted of ioxaglate (negatively charged, q = −1) and bismuth nanoparticles (BiNPs, q = 0, diameter ≈ 0.2 µm). CECT results were compared to histological cartilage proteoglycan content maps acquired using digital densitometry. Results BiNPs enabled prolonged visual detection of both groove types as they are too large to diffuse into the cartilage. Furthermore, proportional ioxaglate diffusion inside the tissue allowed differentiation between the lesion and ungrooved articular cartilage (3 mm from the lesion and contralateral joint). The mean ioxaglate partition in the lesion was 19 percentage points higher (P < 0.001) when compared with the contralateral joint. The digital densitometry and the dual‐contrast CECT findings showed good subjective visual agreement. Main limitations Ex vivo study protocol and a low number of investigated joints. Conclusions The dual‐CECT methodology, used in this study for the first time to image whole equine joints, is capable of effective lesion detection and simultaneous evaluation of the condition of the articular cartilage.


| INTRODUC TI ON
Osteoarthritis (OA) is a frequent joint disease in the horse. 1 The disease may be initiated by joint trauma that produces cartilage lesions, which may further deteriorate and culminate in the development of post-traumatic OA, 2,3 with accompanying clinical signs such as lameness. Early and accurate detection of cartilage damage is key for successful early intervention, allowing for effective treatment and prevention of further damage. 4,5 Animal models provide an important contribution to OA research. 6,7 One of the previously used models is the groove model. This is a model that induces trauma of the hyaline, non-calcified cartilage layer surgically and is used for studying the progression of degenerative changes within a joint. 8,9 Radiography, ultrasonography, arthroscopy, computed tomography (CT) and magnetic resonance imaging (MRI) are medical imaging techniques that have been used for articular cartilage imaging. 10 Magnetic resonance imaging is an excellent imaging modality for imaging soft tissues with high water content, like articular cartilage. However, low magnetic field MRI (≤0.3 tesla), as mostly used in equine practice, cannot detect small cartilage lesions. [11][12][13] Plain radiography and CT allow for faster imaging and significantly better image fidelity for small objects. But, due to relatively similar X-ray attenuation of synovial fluid and cartilage, plain radiography and CT are not able to distinguish cartilage and its lesions from synovial fluid. 10 Contrast agents enhance the distinction between synovial fluid and articular cartilage in MRI and CT images. 14 Anionic (negatively charged) contrast agents, eg ioxaglate, are commonly used for intraarticular contrast-enhanced CT (CECT). 15 The resolution of modern clinical CT devices in combination with contrast media enables accurate evaluation of cartilage thickness and qualitative assessment of cartilage condition. [16][17][18] After the contrast agent injection in vivo, optimal timing allows scanning with maximum deposition of agent inside cartilage, before it clears from the synovial space. The distribution of ioxaglate within cartilage is inversely proportional to the proteoglycan (PG) distribution in the extracellular matrix (related to the negative fixed charge density). 19,20 A superficial collagen disruption, decrease in PG content and the resulting increase in water content are early signs of osteoarthritic degeneration. 21 These changes decrease the fixed charge density and increase the tissue permeability, subsequently increasing the diffusion of negatively charged contrast agents into cartilage. 22 Contrast-enhanced CT with ioxaglate requires two image acquisitions: immediately and at a delayed time point after contrast agent injection. 10,17 The immediate scan evaluates the articulating surface when there is still a good contrast between synovial fluid and articular cartilage. As the contrast agent diffuses into the cartilage layer, this contrast diminishes. The second scan enables quantitative evaluation of the cartilage condition. 17 A bipartite contrast agent, comprising ioxaglate and neutral bismuth nanoparticles (BiNPs) which are large enough to remain in the joint space, allows for characterisation of the tissue using only one scan, without the need for complex co-registration of separate scans. 23 In this study, we aim to investigate the capability of this technique used previously on osteochondral samples to visualise surgically induced blunt and sharp cartilage defects in equine carpi ex vivo. We hypothesise that in addition to visualising the grooves with the dual-contrast technique, we can simultaneously evaluate the PG content of the cartilage.

| Animal selection and preparation
Nine female Shetland ponies (aged 6.8 ± 2.6 years; bodyweight was 203 ± 15.3 kg) were included. As no prior contrast agent (ioxaglate) partition data for whole equine joints were available, the number of ponies was based on a power analysis (power 0.90 and P < 0.05) pertaining to the macroscopic and microscopic scores (OARSI) in previous studies. 8,9,[24][25][26] Retrospectively, based on the ioxaglate partitions as determined in this study, the statistical power was found to range from 0.83 to 0.94, depending on the location and the groove type.
Average cartilage thickness for the ponies was 0.501 ± 0.101 mm (defined previously from micro-CT images at six locations adjacent to grooves). For comparison, the cartilage thicknesses in the carpal joint of trained and untrained Thoroughbred horses has been reported to be 0.7 and 0.6 mm, respectively. 27 Prior to the experiments, the ponies were in good health and did not suffer from clinically visible lameness or joint injuries.

Conclusions:
The dual-CECT methodology, used in this study for the first time to image whole equine joints, is capable of effective lesion detection and simultaneous evaluation of the condition of the articular cartilage.

K E Y W O R D S
articular cartilage, bismuth nanoparticles, contrast-enhanced computed tomography, horse, ioxaglate, osteoarthritis surgical approach to the joint using an incision as small as possible needed for exposure of the articular cartilage (mini-arthrotomy). 25 Blunt or sharp grooves were randomly made in the radial facet of the third carpal bone and in the intermediate carpal bone

| Contrast agent preparation
Bismuth nanoparticles (BiNPs) were prepared according to 'targeted pyrolysis' approach, developed and published by our group. 29

| Contrast-enhanced micro-CT
Joints were thawed overnight at 4°C. Dual-contrast agent was injected into the radiocarpal and middle carpal joints (10 mL each).
After injection, the joints were cyclically flexed and extended for

| Data analysis
To allow visual comparison ( Figure 2) between the two imaging time Brigham and Women's Hospital). 32,33 To reduce noise, the CT images that were used for analysis were Depth-dependent ioxaglate profiles were determined for the lesion, 3 mm away from the groove, and VOI in the contralateral joint.
Statistical significance of differences in ioxaglate partition and optical density (OD) between different locations was evaluated using a Kruskal-Wallis test. The inspection was done separately for the different grooves, but also with the blunt and sharp groups combined.
The level of statistical significance was set at P < 0.05. Bonferroni correction was used to reduce type I error for multiple comparisons.
The statistical analyses were conducted using SPSS (v. 27 SPSS Inc, IBM Company).

| Digital densitometry
After the micro-CT imaging, the grooved sites along with the con- were particularly well visible (Figures 2 and 3), but the thinner sharp grooves were also visually distinguishable on the micro-CT images (Figures 2 and 4). Increased attenuation deeper in the tissue pointed to enhanced ioxaglate penetration and build-up of the contrast agent, as a result of peri-defect deterioration (Figures 3 and 4). This was not observed for healthy cartilage; for the contralateral healthy tissue, the maps of the ioxaglate attenuation displayed homogeneity ( Figure 5).
The resolution of the micro-CT system enabled depth-dependent profiling of the tissue with good accuracy (Figure 6). Mean ioxaglate partition for full thickness cartilage was 19 (P < 0.001) and 16 (P = 0.006) percentage points higher for both types of lesions, compared to the contralateral joint and 3 mm away from the groove, respectively. The same difference between lesion and contralateral joint for the blunt groove was 24 (P = 0.041) percentage points and for the sharp groove was 15 (P = 0.019) percentage points ( Table 1).
OD maps exhibited visual erosion and a decrease in staining (ie estimate of cartilage fixed charge density) at grooved locations ( Figures 3 and 4), similar to the micro-CT images. Significant difference in OD between lesion and contralateral joint was observed for blunt (P = 0.035) and sharp (P = 0.033) grooves ( Table 1). In addition, significant (P = 0.002) difference in OD between the lesion and 3 mm away from the lesion was found for the blunt groove.

| DISCUSS ION
In the current study, delayed dual-contrast micro-CT was used, for the first time, for whole equine joints ex vivo. With a single scan, the method enables simultaneous visualisation of the articulating surfaces and quantification of the tissue degenerative state. We showed that the surgically made grooves can be easily differentiated from the ungrooved cartilage, and the cartilage condition around the groove can be visually evaluated from the differing ioxaglate uptake.
The blunt grooves were clearly distinguishable on the CT images the BiNPs the articulating surface would have been blurry, making segmentation and inspection of the surface condition difficult or impossible. 17 Furthermore, ioxaglate that diffuses into the cartilage does not hinder the visualisation of the interface between the tissue and the joint cavity when combined with the BiNPs (Figure 2). The sharp grooves were visually distinguishable even though OD-maps ( Figure 4) showed that the tissue surface was relatively smooth and fairly regular. te Moller et al reported significant differences between the blunt and sharp grooves in OARSI microscopy scores (P = 0.007) and in fixed charge density (P = 0.006), when cartilage condition around the grooves was inspected. Albeit, the overall degeneration remained moderate. 25 The present dual-contrast CECT method or OD with fixed VOIs were not able to differentiate between the lesion types. In future, use of similar grading scales as introduced in MRI studies (such as the WORMS scoring 35 ) could help with this issue.
The groove model chosen for this study differs from naturally developing idiopathic OA. However, the blunt and sharp grooves have been used to study OA development 8,9,36,37 and these lesions resemble defects that could occur on the cartilage surface after injury, before they lead to post-traumatic OA. In lesion area, cartilage deterioration caused by the blunt and sharp grooves showed as increased X-ray attenuation, as a result of the larger amount of ioxaglate inside the tissue due to the increased permeability and decreased PG content, as confirmed in the OD-maps (Figures 3, 4, 6, and Table 1). This objective, numerical information might benefit surgeons, who need to assess the extent of trauma and decide whether there is a need for surgical intervention. Ioxaglate partition was 60 ± 15% in blunt and sharp grooves, which was significantly  In conclusion, in the current study the dual-contrast method was, for the first time, applied to evaluate the cartilage condition

E TH I C A L A N I M A L R E S E A RCH
The experimental model used in this study was approved by the

Utrecht University Animal Experiments Committee and the Central
Committee for Animal Experiments (permit AVD108002015307).

I N FO R M ED CO N S ENT
Not applicable.

ACK N OWLED G EM ENTS
The authors would like to acknowledge the Equine Clinic of Utrecht University for providing facilities to conduct this study. Eija Rahunen is acknowledged for preparing the histological sections and Dristi Regmi for helping with the digital densitometry measurements.

CO N FLI C T O F I NTE R E S T S
No conflicts of interest have been declared.

PEER R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/evj.13573.